IP3R2 regulates apoptosis by Ca2+ transfer through mitochondria-ER contacts in hypoxic photoreceptor injury.

Mitochondria-associated ER membranes (MAMs) are contact sites that enable bidirectional communication between the ER (endoplasmic reticulum) and mitochondria, including the transfer of Ca2+ signals. MAMs are essential for mitochondrial function and cellular energy metabolism. However, unrestrained Ca2+ transfer to the mitochondria can lead to mitochondria-dependent apoptosis. IP3R2 (Inositol 1,4,5-trisphosphate receptor 2) is an important intracellular Ca2+ channel. This study investigated the contribution of IP3R2-MAMs to hypoxia-induced apoptosis in photoreceptor cells. A photoreceptor hypoxia model was established by subretinal injection of hyaluronic acid (1%) in C57BL/6 mice and 1% O2 treatment in 661W cells. Transmission electron microscopy (TEM), ER-mitochondria colocalization, and the MAM reporter were utilized to evaluate MAM alterations. Cell apoptosis and mitochondrial homeostasis were evaluated using immunofluorescence (IF), flow cytometry, western blotting (WB), and ATP assays. SiRNA transfection was employed to silence IP3R2 in 661W cells. Upon hypoxia induction, MAMs were significantly increased in photoreceptors both in vivo and in vitro. This was accompanied by the activation of mitochondrial apoptosis and disruption of mitochondrial homeostasis. Elevated MAM-enriched IP3R2 protein levels induced by hypoxic injury led to mitochondrial calcium overload and subsequent photoreceptor apoptosis. Notably, IP3R2 knockdown not only improved mitochondrial morphology but also restored mitochondrial function in photoreceptors by limiting MAM formation and thereby attenuating mitochondrial calcium overload under hypoxia. Our results suggest that IP3R2-MAM-mediated mitochondrial calcium overload plays a critical role in mitochondrial dyshomeostasis, ultimately contributing to photoreceptor cell death. Targeting MAM constitutive proteins might provide an option for a therapeutic approach to mitigate photoreceptor death in retinal detachment.

TIN2-mediated reduction of mitophagy induces RPE senescence under high glucose.

The telomere-associated protein TIN2 localizes to both telomeres and mitochondria. Nevertheless, the impact of TIN2 on retinal pigment epithelial (RPE) cells in diabetic retinopathy (DR) remains unclear. This research aims to examine the role of TIN2 in the senescence of RPE and its potential as a therapeutic target. Western blotting and immunofluorescence staining were utilized to identify TIN2 expression and mitophagy. RT-qPCR was employed to identify senescent associated secretory phenotype (SASP) in ARPE-19 cells infected with TIN2 overexpression. To examine mitochondria and the cellular senescence of RPE, TEM, SA-ß-gal staining, and cell cycle analysis were used. The impact of TIN2 was examined using OCT and immunohistochemistry in mice. DHE staining and ZO-1 immunofluorescence were applied to detect RPE oxidative stress and tight junctions. Our research revealed that increased mitochondria-localized TIN2 aggravated the cellular senescence of RPE cells both in vivo and in vitro under hyperglycemia. TIN2 overexpression stimulated the mTOR signaling pathway in ARPE-19 cells and exacerbated the inhibition of mitophagy levels under high glucose, which can be remedied through the mTOR inhibitor, rapamycin. Knockdown of TIN2 significantly reduced senescence and mitochondrial oxidative stress in ARPE-19 cells under high glucose and restored retinal thickness and RPE cell tight junctions in DR mice. Our study indicates that increased mitochondria-localized TIN2 induced cellular senescence in RPE via compromised mitophagy and activated mTOR signaling. These results propose that targeting TIN2 could potentially serve as a therapeutic strategy in the treatment of DR.

UCP2-SIRT3 Signaling Relieved Hyperglycemia-Induced Oxidative Stress and Senescence in Diabetic Retinopathy.

Purpose: Diabetic retinopathy (DR) is one of the most common reasons for blindness. uncoupling protein 2 (UCP2), an uncoupling protein located in mitochondria, has been reported to be related to metabolic and vascular diseases. This research aimed to illustrate the function and mechanism of UCP2 in the pathogenesis of DR. Methods: Human epiretinal membranes were collected to investigate the expression of UCP2 by quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence. Primary human retinal microvascular endothelial cells (HRECs) were cultured in high glucose (HG) to establish an in vitro cell model for DR. Flow cytometry analysis was used to measure intracellular reactive oxygen species (ROS). Senescence levels were evaluated by the senescence-associated beta-galactosidase (SA-ß-gal) assay, the expression of senescence marker P21, and cell-cycle analysis. Adenovirus-mediated UCP2 overexpression or knockdown and specific inhibitors were administered to investigate the underlying regulatory mechanism. Results: Proliferative fibrovascular membranes from patients with DR illustrated the downregulation of UCP2 and sirtuin 3 (SIRT3) by qRT-PCR and immunofluorescence. Persistent hyperglycemia-induced UCP2 downregulation in the progress of DR and adenovirus-mediated UCP2 overexpression protected endothelial cells from hyperglycemia-induced oxidative stress and senescence. Under hyperglycemic conditions, UCP2 overexpression attenuated NAD+ downregulation; hence, it promoted the expression and activity of SIRT3, an NAD+-dependent deacetylase regulating mitochondrial function. 3-TYP, a selective SIRT3 inhibitor, abolished the UCP2-mediated protective effect against oxidative stress and senescence. Conclusions: UCP2 overexpression relieved oxidative stress and senescence based on a novel mechanism whereby UCP2 can regulate the NAD+-SIRT3 axis. Targeting oxidative stress and senescence amelioration, UCP2-SIRT3 signaling may serve as a method for the prevention and treatment of DR and other diabetic vascular diseases.

TRIM25 inhibition attenuates inflammation, senescence, and oxidative stress in microvascular endothelial cells induced by hyperglycemia.

PURPOSES: This work aimed to assess the possible role of TRIM25 in regulating hyperglycemia-induced inflammation, senescence, and oxidative stress in retinal microvascular endothelial cells, all of which exert critical roles in the pathological process of diabetic retinopathy. METHODS: The effects of TRIM25 were investigated using streptozotocin-induced diabetic mice, human primary retinal microvascular endothelial cells cultured in high glucose, and adenoviruses for TRIM25 knockdown and overexpression. TRIM25 expression was evaluated by western blot and immunofluorescence staining. Inflammatory cytokines were detected by western blot and quantitative real-time PCR. Cellular senescence level was assessed by detecting senescent marker p21 and senescence-associated-ß-galactosidase activity. The oxidative stress state was accessed by detecting reactive oxygen species and mitochondrial superoxide dismutase. RESULTS: TRIM25 expression is elevated in the endothelial cells of the retinal fibrovascular membrane from diabetic patients compared with that of the macular epiretinal membrane from non-diabetic patients. Moreover, we have also observed a significant increase in TRIM25 expression in diabetic mouse retina and retinal microvascular endothelial cells under hyperglycemia. TRIM25 knockdown suppressed hyperglycemia-induced inflammation, senescence, and oxidative stress in human primary retinal microvascular endothelial cells while TRIM25 overexpression further aggregates those injuries. Further investigation revealed that TRIM25 promoted the inflammatory responses mediated by the TNF-α/NF-κB pathway and TRIM25 knockdown improved cellular senescence by increasing SIRT3. However, TRIM25 knockdown alleviated the oxidative stress independent of both SIRT3 and mitochondrial biogenesis. CONCLUSION: Our study proposed TRIM25 as a potential therapeutic target for the protection of microvascular function during the progression of diabetic retinopathy.

SYVN1 Promotes STAT3 Protein Ubiquitination and Exerts Antiangiogenesis Effects in Retinopathy of Prematurity Development.

Purpose: SYVN1, a gene involved in endoplasmic reticulum-associated degradation, has been found to exert a protective effect by inhibiting inflammation in retinopathy. This study aimed to clarify whether SYVN1 is involved in the pathogenesis of retinopathy of prematurity (ROP) and its potential as a candidate for target therapy. Methods: Human retinal microvascular endothelial cells (hRMECs) and a mouse model of oxygen-induced retinopathy (OIR) were used to reveal the retinopathy development-associated protein expression and molecular mechanism. An adenovirus overexpressing SYVN1 or vehicle control was injected intravitreally at postnatal day 12 (P12), and the neovascular lesions were evaluated in retinal flatmounts with immunofluorescence staining, and hematoxylin and eosin staining at P17. Visual function was assessed by using electroretinogram (ERG). Results: Endogenous SYVN1 expression dramatically decreased in hRMECs under hypoxia and in ROP mouse retinas. SYVN1 regulated the signal transducer and activator of transcription 3 (STAT3)/vascular endothelial growth factor (VEGF) axis. SYVN1 overexpression promoted ubiquitination and degradation of STAT3, decreased the levels of phospho-STAT3, secretion of VEGF, and formation of neovascularization in hRMECs, which could be rescued by STAT3 activator treatment. In addition, SYVN1 overexpression prevented neovascularization and extended physiologic retinal vascular development in the retinal tissues of OIR mice without affecting retinal function. Conclusions: SYVN1 has a protective effect against OIR, and the molecular mechanisms are partly through SYVN1-mediated ubiquitination of STAT3 and the subsequent downregulation of VEGF. These findings strongly support our assumption that SYVN1 confers ROP resistance and may be a potentially novel pharmaceutical target against proliferative retinopathy.

Enhancement of glycolysis-dependent DNA repair regulated by FOXO1 knockdown via PFKFB3 attenuates hyperglycemia-induced endothelial oxidative stress injury.

The accumulation of DNA damage induced by oxidative stress is a crucial pathogenic factor of endothelial loss in diabetic vascular complications, but it is still unknown whether aberrant glucose metabolism leads to defective DNA repair and accounts for hyperglycemia-induced endothelial oxidative stress injury. Here, we showed that Foxo1 knockdown alleviated diabetes-associated retinal DNA damage and vascular dysfunction. Mechanistically, FOXO1 knockdown avoided persistent DNA damage and cellular senescence under high glucose in endothelial cells by promoting DNA repair mediated by the MRN (MRE11-RAD50-NBS1 complex)-ATM pathway in response to oxidative stress injury. Moreover, FOXO1 knockdown mediated robust DNA repair by restoring glycolysis capacity under high glucose. During this process, the key glycolytic enzyme PFKFB3 was stimulated and, in addition to its promoting effect on glycolysis, directly participated in DNA repair. Under genotoxic stress, PFKFB3 relocated into oxidative stress-induced DNA damage sites and promoted DNA repair by interaction with the MRN-ATM pathway. Our study proposed that defective glycolysis-dependent DNA repair is present in diabetic endothelial cells and contributes to hyperglycemia-induced vascular dysfunction, which could provide novel therapeutic targets for diabetic vascular complications.

FBXW7 alleviates hyperglycemia-induced endothelial oxidative stress injury via ROS and PARP inhibition.

Diabetic retinopathy (DR) and other diabetic vascular complications are the leading cause of death and disability in patients with suboptimum glycemic control. In the pathogenesis of diabetic vascular diseases, hyperglycemia-induced oxidative stress, DNA damage, and poly-ADP-ribose-polymerase (PARP) hyperactivation play important roles in endothelial cell impairment. Adipose differentiation-related protein FBXW7 was reported to regulate PGC-1α stability and mitochondrial homeostasis. Here, we investigated the role and mechanism of FBXW7 in repairing endothelial oxidative stress injuries under hyperglycemic conditions. FBXW7 promoted the hampered activity of homologous recombination and non-homologues end joining pathway for repairing DNA double-strand breaks damage, an initiating factor for PARP hyperactivation and diabetic vascular complications. The abundant mobilization of DNA damage repair mediated by FBXW7 suppressed PARP activation, leading to downregulation of PARP expression and activity in both human endothelial cells and diabetic rat retinas. This provided a new method for PARP inhibition, superior to PARP inhibitors for treating diabetic vascular complication. Furthermore, FBXW7 rescued downregulated NAD+ levels and ameliorated mitochondrial dysfunction, thereby reducing superoxide production under hyperglycemic conditions. These effects reversed oxidative injury and vascular leakage in diabetic rat retina, providing a potential future treatment strategy.

Discrimination of Temperature and Strain in Brillouin Optical Time Domain Analysis Using a Multicore Optical Fiber.

Brillouin optical time domain analysis is the sensing of temperature and strain changes along an optical fiber by measuring the frequency shift changes of Brillouin backscattering. Because frequency shift changes are a linear combination of temperature and strain changes, their discrimination is a challenge. Here, a multicore optical fiber that has two cores is fabricated. The differences between the cores' temperature and strain coefficients are such that temperature (strain) changes can be discriminated with error amplification factors of 4.57 °C/MHz (69.11 µ ϵ /MHz), which is 2.63 (3.67) times lower than previously demonstrated. As proof of principle, using the multicore optical fiber and a commercial Brillouin optical time domain analyzer, the temperature (strain) changes of a thermally expanding metal cylinder are discriminated with an error of 0.24% (3.7%).

All-optical Brillouin dynamic grating generation in few-mode optical fiber.

We report, for the first time (to our knowledge), generation of Brillouin dynamic gratings (BDGs) in few-mode optical fibers. A moving acoustic grating is generated by stimulated Brillouin scattering using a writing beam in one fiber mode, which is used to reflect a reading beam at a different wavelength in another fiber mode. With single-end pumping, a BDG with a tunable reflectance of up to 0.1% is demonstrated in a 15 km specially designed two-mode optical fiber.

Compensation-free, all-fiber-optic, two-photon endomicroscopy at 1.55 µm.

We present an all-fiber-optic scanning multiphoton endomicroscope with 1.55 µm excitation without the need for prechirping femtosecond pulses before the endomicroscope. The system consists of a 1.55 µm femtosecond fiber laser, a customized double-clad fiber for light delivery and fluorescence collection, and a piezoelectric scan head. We demonstrate two-photon imaging of cultured cells and mouse tissue, both labeled with indocyanine green. Free-space multiphoton imaging with near-IR emission has previously shown benefits in reduced background fluorescence and lower attenuation for the fluorescence emission. For fiber-optic multiphoton imaging there is the additional advantage of using the soliton effect at the telecommunication wavelengths (1.3-1.6 µm) in fibers, permitting dispersion-compensation-free, small-footprint systems. We expect these advantages will help transition multiphoton endomicroscopy to the clinic.

Wavelength tunable stretched-pulse mode-locked all-fiber erbium ring laser with single polarization fiber.

A wavelength tunable stretched-pulse mode-locked all-fiber ring laser using single polarization fiber (SPF) was demonstrated. In this laser, a segment of SPF was used simultaneously as a polarizer and a tunable filter in the laser cavity. Self-starting mode-locking with femtosecond output pulses was demonstrated. A wavelength tuning of ~20nm was achieved by bending the SPF with different radii.